Summary

Vertebrate oocytes are maintained in meiotic arrest for prolonged periods
of time before undergoing oocyte maturation in preparation for fertilization.
Cyclic AMP (cAMP) signaling plays a crucial role in maintaining meiotic
arrest, which is released by a species-specific hormonal signal. Evidence in
both frog and mouse argues that meiotic arrest is maintained by a
constitutively active G-protein coupled receptor (GPCR) leading to high cAMP
levels. Because activated GPCRs are typically targeted for endocytosis as part
of the signal desensitization pathway, we were interested in determining the
role of trafficking at the cell membrane in maintaining meiotic arrest. Here
we show that blocking exocytosis, using a dominant-negative SNAP25 mutant in
Xenopus oocytes, releases meiotic arrest independently of
progesterone. Oocyte maturation in response to the exocytic block induces the
MAPK and Cdc25C signaling cascades, leading to MPF activation, germinal
vesicle breakdown and arrest at metaphase of meiosis II with a normal bipolar
spindle. It thus replicates all tested aspects of physiological maturation.
Furthermore, inhibiting clathrin-mediated endocytosis hinders the
effectiveness of progesterone in releasing meiotic arrest. These data show
that vesicular traffic at the cell membrane is crucial in maintaining meiotic
arrest in vertebrates, and support the argument for active recycling of a
constitutively active GPCR at the cell membrane.

Given the fact that most cells have developed complex cascades to limit and
regulate GPCR signaling, it is intriguing that oocyte meiotic arrest is
dependent on extended constitutive GPCR signaling. This raises the interesting
question of the mechanisms involved in allowing such prolonged GPCR signaling
in oocytes. An important pathway for GPCR desensitization is through endocytic
removal of the receptor from the cell membrane following GRK phosphorylation
and arrestin binding (Moore et al.,
2006). If this pathway is indeed functional in oocytes, then these
cells must have developed mechanisms to replenish active GPCRs at the cell
membrane to maintain meiotic arrest. Here we explore the role of vesicular
trafficking at the cell membrane in maintaining meiotic arrest in
Xenopus oocytes. We show that meiotic arrest requires a functional
exocytic pathway, as blocking exocytosis with a dominant-negative SNAP25
(SNAP25Δ20) releases meiotic arrest in the absence of the physiological
stimulus, progesterone. The effect of SNAP25Δ20 expression on
trafficking at the cell membrane was followed by measuring membrane
capacitance, which provides a direct measure of membrane area and has been
shown to closely track the trafficking pattern of several membrane proteins in
Xenopus oocytes (Peters et al.,
1999; Quick et al.,
1997; Awayda,
2000). SNAP25Δ20-induced maturation is normal in every
aspect tested: it induces the MAPK and Cdc25C cascades leading to MPF
activation, germinal vesicle breakdown (GVBD) and arrest at metaphase II of
meiosis with a normal bipolar spindle. Furthermore, blocking clathrin-mediated
endocytosis hinders the effectiveness of saturating levels of progesterone in
releasing oocytes from meiotic arrest. Together, these data show that
vesicular trafficking at the cell membrane is a crucial determinant of meiotic
arrest.

MATERIALS AND METHODS

Gamete preparation and treatments

Xenopus oocytes were obtained as previously described
(Machaca and Haun, 2002).
Oocyte maturation was induced with 5 μg/ml progesterone. GVBD was detected
visually by the appearance of a white spot at the animal pole and confirmed by
fixing oocytes in methanol and bisecting them in half to visualize the
germinal vesicle (see Fig. 2B).
For forskolin and cholera toxin treatments, oocytes were pre-incubated in 100μ
M forskolin or 5×10-9 M cholera toxin (Calbiochem) for
30-60 minutes before progesterone treatment, whereas SNAP25Δ20-injected
cells were treated with the drugs at the time of injection.

Capacitance measurement

Oocytes were voltage clamped with two microelectrodes by the use of a
GeneClamp 500 (Axon Instruments). Electrodes were filled with 3 M KCl and had
resistances of 0.5-2 MΩ. Oocytes were bathed in Ringer, in mM: 96 NaCl,
2.5 KCl, 1.8 CaCl2, 2 MgCl2, 10 HEPES, pH 7.4. Voltage
stimulation and data acquisition were controlled using pClamp8 (Axon
Instruments). Membrane capacitance (Cm) was measured using the built-in
algorithm in pClamp8 with a voltage pulse of 5 mV. This algorithm accurately
reproduced capacitance values obtained by direct calculation as previously
described (Machaca and Haun,
2000). Specifically, four steps from a -35 mV holding potential to
-30 mV for 50 msec each were administered. Capacitive current decay was
averaged and fitted by a single exponential. Membrane capacitance
Cm was calculated as τ (1/Ra+Gm). τ
is the time constant obtained from the exponential fit. Ra is the
access resistance and was calculated as Vp/I0.
Vp is the applied voltage pulse (5 mV) and I0 is the
instantaneous current obtained by extrapolating the experimental fit to time
0. Gm was calculated as
Iss/(Vp-Ra*Iss).
Iss is the steady state current following relaxation of the
capacitive transient (Takahashi et al.,
1996).

Molecular biology

For the wild-type full-length SNAP25, mouse Snap25B in pcDNA3
(Low et al., 1998) was
subcloned into the BamHI-XhoI sites in the Xenopus
oocyte expression vector pSGEM, which flanks the cDNA with the 5′- and
3′-UTRs from the Xenopus globin gene, thus stabilizing the
resultant mRNA in the oocyte (Liman et
al., 1992). The mouse Snap25 gene was highly likely to be
functional in Xenopus, as it shares 95% identity with
Xenopus SNAP25. SNAP25Δ20 was generated from pcDNA3-SNAP25 by
PCR using a pair of primers that flanked the clone with
BamHI-EcoRI sites for subcloning into pSGEM, and that
introduced a stop codon after residue 186, thus deleting the last 20 residues.
mRNAs for the SNAP25 clones in pSGEM were produced by in vitro transcription
after linearizing the vector with NheI using the mMessage mMachine T7
kit (Ambion). The Mos clone was previously described
(Machaca and Haun, 2002).

Transferrin endocytosis assay

Cells were treated overnight with 332 μM monodansylcadaverine (MDC)
(Sigma) or the carrier control (DMSO), washed in OR2 (82.5 mM NaCl, 2.5 mM
KCl, 1 mM Na2HPO4, 1 mM CaCl2, 5 mM HEPES, pH
7.5) for 5 minutes and incubated OR2 containing Alexa-fluor-633-conjugated
transferrin at a concentration of (125 μg/ml) for 15 minutes. Then they
were rinsed extensively in OR2, and the extent of transferrin internalization
at the vegetal pole was imaged on a Zeiss LSM510 confocal microscope
(10× objective). Cross sections at several planes across the oocytes
were scanned and showed consistent levels of transferrin internalization.
Therefore, data were collected from a single plane at an equivalent depth into
the vegetal hemisphere. Images were thresholded and subjected to morphometry
analysis using the MetaMorph software. This allowed quantification of the
number of early endosomes, their equivalent sphere volume, and average and
total transferrin fluorescent intensity.

Imaging spindle structure

Cells were fixed 3 hours after GVBD in 100% methanol and stored at
-20°C overnight. After rehydration in TBS:methanol (1:1) for 20 minutes,
oocytes were washed twice with TBS for 15 minutes each and blocked for 3 hours
in TBS containing 2% BSA. Oocytes were then immunolabeled with an
anti-α-tubulin monoclonal antibody (DMA1, Sigma) in TBS containing 2%
BSA, followed by a Cy2-conjugated donkey anti-mouse secondary (Jackson) for 24
hours each. The oocytes were washed five times in TBS for 24 hours and stained
with 1 μM Sytox Orange (Molecular Probes). After staining cells were washed
in TBS for 1 hour, dehydrated in 100% methanol for 30 minutes and cleared in
benzyl alcohol/benzyl benzoate (1:2). Spindle structure images were collected
on a Zeiss LSM510 confocal microscope (20× objective).

RESULTS

SNAP25Δ20 blocks exocytosis

SNARE proteins are central to vesicular fusion events that underlie
trafficking at the subcellular level (Chen
and Scheller, 2001). SNAREs contain coiled-coil domains that
interact to form a four-helix bundle, which is important for vesicle fusion.
Three helices are contributed by Q-SNAREs present on one membrane, and the
fourth helix is contributed by an R-SNARE on the other membrane
(Salaun et al., 2004). In the
case of exocytosis, two of the four helices that form the four-helix bundle
are contributed by SNAP25 (Jahn,
2004). SNAP25 contains two coiled-coil domains at its N- and
C-termini and a central cysteine-rich domain that mediates membrane
localization through palmitoylation
(Salaun et al., 2004). Because
all four SNARE helices are required for membrane fusion, deletion of a SNAP25
coiled-coil domain is predicted to block exocytosis. Indeed, a SNAP25 mutant
that removes the last 20 residues (SNAP25Δ20) has been reported to act
as a dominant-negative of exocytosis in Xenopus oocytes
(Yao et al., 1999). Therefore,
to test the role of exocytosis in maintaining meiotic arrest we constructed a
SNAP25Δ20 clone and expressed it in oocytes
(Fig. 1). By recording membrane
capacitance as a direct measure of cell membrane area, we functionally
confirmed that SNAP25Δ20 acts as a dominant-negative and effectively
blocks exocytosis (Fig. 1).
SNAP25Δ20, but not full-length wild-type SNAP25, decreased membrane area
in a time-dependent fashion (Fig.
1). The expression levels of both SNAP25Δ20 and wild-type
SNAP25 were comparable (Fig.
1).

Dominant-negative SNAP25Δ20 blocks exocytosis.Xenopus ocytes were injected with either wild-type or SNAP25Δ20
mRNA (5-10 ng) and membrane capacitance was measured [Cm(nF)] using two
electrode-voltage clamp over time, as indicated (mean±s.e.;
n=8-20). Western blot analysis using SNAP25 antibody shows similar
levels of expression of wild-type and SNAP25Δ20, and no band was
detected in uninjected oocytes. SNAP25Δ20 runs faster than wild-type
full-length SNAP25 because of the deletion of the last 20 residues. Ooc,
uninjected oocytes; WT, wild type.

SNAP25Δ20 induces oocyte maturation

Expression of SNAP25Δ20 induced progesterone-independent oocyte
maturation (Fig. 2). Oocytes
expressing SNAP25Δ20 entered meiosis, as marked by GVBD, with the same
efficiency as progesterone-treated oocytes
(Fig. 2A). By contrast, oocytes
injected with wild-type SNAP25 mRNA did not mature
(Fig. 2A).
SNAP25Δ20-dependent oocyte maturation was associated with the appearance
of a normal white spot on the animal hemisphere, and with the expected
breakdown of the nuclear envelope (Fig.
2B). Furthermore, SNAP25Δ20-injected oocytes extruded a
polar body, showing that they completed meiosis I, and arrested at metaphase
of meiosis II with a normal metaphase II spindle
(Fig. 2C).

By assessing the time required for 50% of the oocytes in the population to
reach GVBD (G50), one can obtain a measure of the rate at which
oocytes mature. SNAP25Δ20-induced maturation occurred with significantly
slower kinetics compared with progesterone
(Fig. 2D). This is not
surprising given the time required for translation of the injected mRNA before
a functional block of exocytosis can be achieved.

Oocyte maturation is ultimately dependent on the activation of
maturation-promoting factor (MPF/cdk1-cyclin B), which is the primary activity
that regulates G2/M transition in both mitosis and meiosis, and consists of a
catalytic p34cdc2 Ser/Thr kinase subunit and a regulatory cyclin B
subunit (Coleman and Dunphy,
1994). Two signaling cascades combine to induce the dramatic
activation of MPF at GVBD: the MAPK-cascade, which leads to inhibition of the
MPF-inhibitory kinase Myt1 (Palmer et al.,
1998; Nebreda and Ferby,
2000), and the polo-like kinase-Cdc25C cascade, which activates
Cdc25C. Cdc25C is a dual-specificity phosphatase that removes the inhibitory
phosphorylation at Tyr15 and Thr14 from cdc2 kinase, and constitutes the
rate-limiting step in MPF activation
(Perdiguero and Nebreda, 2004;
Kumagai and Dunphy, 1991).

To determine whether the signaling cascade underlying oocyte maturation is
activated normally in SNAP25Δ20-injected cells, we measured the
activation of MAPK and MPF (Fig.
2E). For these experiments, oocyte lysates were prepared at
different time points during maturation: (1) when the first cells in the
population reached GVBD (GVBD); (2) when 50% of the cells reached GVBD
[G50; for this time point, lysates from oocytes with a white spot
(w) and those without (nw) were collected]; (3) when 100% of the cells in the
population reached GVBD (G100). SNAP25Δ20 induced similar
activation profiles as progesterone for both MAPK and MPF, supporting the
argument that it induces oocyte maturation using the same pathways activated
by the physiological hormone (Fig.
2E). In a similar fashion, SNAP25Δ20 induced Cdc25C
activation, as marked by its supershift due to hyperphosphorylation
(Fig. 2F). The wild-type SNAP25
control did not activate MAPK, MPF (Fig.
2E) or Cdc25C (Fig.
2F), although it was typically expressed at higher levels than
SNAP25Δ20 (Fig. 2E,F).
Together, these results show that SNAP25Δ20 induces normal oocyte
maturation at the biochemical, morphological and nuclear maturation (meiosis)
levels.

Timecourse of SNAP25Δ20-dependent maturation

We then analyzed the timecourse of SNAP25Δ20-induced maturation in
more detail to determine if it is equivalent to progesterone treatment.
Oocytes were either injected with SNAP25Δ20 mRNA or treated with
progesterone, and GVBD and capacitance were measured over time. In addition,
lysates were collected for analysis of kinase activation and SNAP25Δ20
expression. After progesterone treatment, membrane area decreases gradually
over time, as previously reported (Kado et
al., 1981; Machaca and Haun,
2000). Oocytes begin to undergo GVBD when capacitance in the
population reaches ∼165 nF, and capacitance continues to decrease as
maturation progresses (Fig. 3A)
(Machaca and Haun, 2000). MAPK
is phosphorylated ∼2.5 hours before GVBD, and MPF activation as marked by
cdc2 dephosphorylation, does not occur until the GVBD stage
(Fig. 3B). The kinetics of
kinase activation and capacitance decrease in SNAP25Δ20-injected cells
is similar (Fig. 3). MAPK
activates ∼1.5 hours before GVBD, and MPF activates at the GVBD stage
(Fig. 3B). Membrane capacitance
in SNAP25Δ20-injected cells reaches ∼135 nF when cells begin to
undergo GVBD (Fig. 3A). Most
interesting is the expression of SNAP25Δ20 protein, which is first
detectable ∼2 hours post-RNA injection, and accumulates to significant
levels ∼5 hours after RNA injection
(Fig. 3B). This 2-5 hour delay
in expression of SNAP25Δ20 explains the delay in GVBD kinetics
(Fig. 3A). These data support
the argument that once SNAP25Δ20 is expressed at high enough levels to
induce a significant block in exocytosis, as measured by membrane capacitance
(Fig. 3A), it is capable of
inducing oocyte maturation with similar kinetics to progesterone. These
results raise the intriguing possibility that a block of exocytosis is a
functionally important component of progesterone-induced maturation.

Botulinum neurotoxin (BoNT) acts synergistically with
progesterone

To corroborate the results of the dominant-negative SNAP25Δ20, we
tested the effects on maturation of BoNT A, a zinc-dependent protease that
cleaves and inactivates SNAP25 (Sudhof,
1995). Injection of various amounts (100-600 nM) of the
catalytically active light chain of BoNT A was insufficient to induce oocyte
maturation. However, when BoNT A-injected oocytes
(Fig. 3C, BoNTA 200 nM) were
treated with sub-threshold levels of progesterone (100 nM), they activated
fully, with similar kinetics to oocytes matured using supra-maximal
progesterone (Fig. 3C, Prog).
By contrast, control BSA-injected oocytes exhibited low levels of maturation
at sub-threshold progesterone (Fig.
3C, BSA). This synergistic effect of BoNT A at sub-threshold
levels of progesterone was observed in 2/4 experiments on oocytes from
different donor females, showing that BoNT A is only mildly effective at
blocking exocytosis. Indeed, in contrast to the robust exocytic block with
SNAP25Δ20, which effectively reduces membrane capacitance
(Fig. 1), no effect of BoNT A
injection on membrane capacitance could be detected
(Fig. 3D). However, the
synergistic effect of BoNT A supports the argument that BoNT A blocks
exocytosis sufficiently to potentiate the effects of sub-threshold levels of
progesterone (Fig. 3C). This
suggests that the exocytic block is a physiological mechanism mediating
progesterone action. Therefore, the fact that BoNT A can potentiate the
ability of sub-threshold progesterone to induce maturation supports our
results with the dominant-negative SNAP25Δ20-dependent exocytic
block.

SNAP25Δ20 releases oocyte meiotic arrest in
Xenopus. (A) Percentage of oocytes reaching the GVBD stage
following progesterone treatment, or injection of SNAP25Δ20 or wild-type
SNAP25 mRNA (mean±s.e.; n=4-7 experiments with >50 oocytes
in each treatment). The percentages reported are the maximal levels of GVBD
reached. (B) Photographs showing the absence of a white spot on the
animal hemisphere, and the presence of the germinal vesicle (nucleus) (arrow)
in untreated oocytes (Ooc) and oocytes injected with wild-type SNAP25 mRNA
(WT). By contrast, progesterone (Prog) or SNAP25Δ20 (Δ20)
injection results in the appearance of a white spot on the animal pole and
GVBD. Top row, white spot; bottom row, GVBD. (C) Spindle structure
(top) and polar body (bottom) in progesterone-treated and
SNAP25Δ20-injected oocytes. Spindle structure was visualized by indirect
immunofluorescence using an anti-tubulin antibody and chromosomes were stained
with Sytox Orange (scale bars: 5 μm for spindles and 10 μm for polar
body). (D) Time required for 50% of the oocytes in the population to
reach the GVBD stage of maturation (GVBD50) following progesterone
treatment or SNAP25Δ20 mRNA injection (mean±s.e.; n=7
experiments from different female donors). (E) Western blot analysis of
cells treated with progesterone or injected with either SNAP25Δ20 or
SNAP25 wild-type mRNA. Lysates were prepared at different time points during
oocyte maturation: (1) untreated oocytes; (2) when oocytes first reached the
GVBD stage (GVBD); (3) when 50% of the cells reach GVBD (G50). In
this case, lysates were prepared from cells with (w) and without (nw) a white
spot; (4) when 100% of the cells reached the GVBD stage (G100).
Because oocytes injected with wild-type SNAP25 mRNA do not undergo GVBD,
lysates were prepared at the same time points when SNAP25Δ20-injected
cells reached the G50 and G100 milestones, indicated as
G50eq and G100eq, respectively. Blots were probed with
anti-phospho-MAPK, anti-phospho-cdc2 and anti-SNAP25 antibodies. (F)
Lysates from oocytes that have undergone GVBD at the G50 time point
were prepared and western blot analysis performed to assess Cdc25C activation
and SNAP25 expression. Cdc25C activation was detected as a supershift on the
gel due to hyperphosphorylation (arrowheads) from the basal state (arrow)
observed in oocytes. In contrast to progesterone or SNAP25Δ20, no shift
is detected in oocytes or SNAP25 wild-type injected cells. The middle band is
a non-specific band. Blots were stripped and re-probed with anti-SNAP25
antibody (lower panel).

Analysis of SNAP25Δ20 site of action

We were then interested in mapping the site of action of SNAP25Δ20 in
releasing meiotic arrest along the physiological oocyte maturation cascade.
Because SNAP25 acts specifically at the cell membrane, it is expected that
inhibition of early steps known to be involved in oocyte maturation should
block SNAP25Δ20-mediated maturation. As discussed above, these include a
block of AC through a G-protein-dependent pathway, leading to a decrease in
cAMP levels. We therefore tested the effects of agents that maintain cAMP
levels high in the oocyte on SNAP25Δ20-mediated maturation
(Fig. 4). Forskolin activates
AC and has been shown to block progesterone-dependent oocyte maturation
(Schorderet-Slatkine and Baulieu,
1982). Indeed, forskolin blocks both progesterone- and
SNAP25Δ20-mediated maturation, showing that both act upstream of AC
(Fig. 4A). As expected,
forskolin blocks the activation of both MAPK and MPF compared with control
cells, and importantly forskolin does not significantly inhibit
SNAP25Δ20 protein expression levels
(Fig. 4A). For these analyses
it is important to confirm that factors that are known to act downstream of
the step of interest are capable of rescuing the block. This is particularly
the case for cAMP, as PKA could have multiple direct effects on downstream
effectors crucial for oocyte maturation. For example, cdc25 has been
identified as a target for PKA, leading to its inhibition
(Duckworth et al., 2002).
Therefore, it is possible that high levels of PKA block maturation at later
steps along the oocyte maturation cascade, such as cdc25. To rule out this
possibility we confirmed that the forskolin block could be rescued by Mos
injection to directly activate the MAPK cascade
(Fig. 4C). These results show
that SNAP25Δ20 acts upstream of AC to release oocyte meiotic arrest.

Timecourse analysis of SNAP25Δ20-induced oocyte maturation
in Xenopus. (A) Extent of GVBD (black) and membrane
capacitance (blue) in progesterone- (left panel) and SNAP25Δ20- (right
panel) treated cells. The inset (below left) shows no change in capacitance or
maturation in untreated oocytes over a similar timecourse. (B) MAPK and
MPF activation and SNAP25 expression over the maturation timecourse following
SNAP25Δ20 mRNA injection (left panel) and progesterone treatment (right
panel). (C) Oocytes were either left untreated and matured with maximal
levels of progesterone (16 μM), or injected with the light chain of BoNT A
(200 nM) or BSA as the carrier control and matured with sub-threshold levels
of progesterone (100 nM). The maturation timecourses for the three treatments
show that BoNT A injection potentiates the effects of sub-threshold levels of
progesterone on maturation. This experiment was repeated four times on oocytes
from different donor females, and the potentiation effects of BoNT A were
observed in 2/4 experiments. (D) Membrane capacitance (Cm)
measurements at different times, as indicated, after BSA or BoNT A (200 nM)
injection, showing that BoNT A is ineffective at reducing Cm.

We also tested the effect of cholera toxin, which ADP-ribosylates
Gαs and activates it, leading to increased activation of AC
and a rise in cAMP levels (Gill and Meren,
1978). Similarly to forskolin, cholera toxin blocks both
progesterone- and SNAP25Δ20-mediated maturation, without dramatically
affecting SNAP25Δ20 protein expression levels
(Fig. 4B). However, in contrast
to forskolin, Mos RNA injection was ineffective at rescuing the cholera
toxin-mediated inhibition (Fig.
4C), supporting the argument that cholera toxin - which is likely
to be a more potent activator of AC, as it catalytically activates
Gαs - inhibits maturation by acting both at the early steps
and later steps downstream of Mos during maturation. Nonetheless, the
forskolin results confirm that SNAP25Δ20 acts upstream of AC, consistent
with the functional role of SNAP25 at the cell membrane.

Role of endocytosis in maintaining meiotic arrest

If as predicted by the exocytosis block data, the residence time of a
constitutive G-protein coupled receptor at the plasma membrane ultimately
modulates meiotic arrest, a block of endocytosis is expected to negatively
modulate oocyte maturation. Specifically, clathrin-dependent endocytosis would
be of primary interest, because it is the predominant internalization pathway
for activated GPCRs (Moore et al.,
2006). To test the role of endocytosis in meiotic arrest, we
inhibited both constitutive and clathrin-mediated endocytosis
(Fig. 5). Constitutive
endocytosis in Xenopus oocytes, the housekeeping pathway that
maintains plasma membrane homeostasis, can be inhibited using Clostridium
botulinum C3 exoenzyme, which ADP-ribosylates and inactivates RhoA
(Schmalzing et al., 1995).
Indeed, injection of oocytes with C3 exoenzyme results in an increase in
membrane capacitance consistent with an endocytic block
(Fig. 5A). However, blocking
constitutive endocytosis did not affect the rate or extent of oocyte
maturation (Fig. 5B), arguing
that the constitutive endocytic pathway is not involved in regulating meiotic
arrest.

Activation of adenylate cyclase inhibits SNAP25Δ20-induced
maturation. (A,B) Extent of maturation of Xenopus
oocytes treated with progesterone or injected with SNAP25Δ20 mRNA in the
presence or absence of 100 μM forskolin (A) or 5×10-9 M
cholera toxin (B). Timecourses of maturation were carried out and the plateau
levels of GVBD at the end of the experiments are reported (above). The
activation of MAPK and MPF and the expression level of SNAP25Δ20 are
also shown (below). Lysates for western blot analysis were prepared from cells
at the GVBD50 time point. (C) Percent GVBD of Mos mRNA- (10
ng) injected oocytes in the presence or absence of 100 μM forskolin or
5×10-9 M cholera toxin. The inset (below) shows the time to
GVBD50 for Mos-injected cells±forskolin. CT, cholera toxin;
Forsk, forskolin.

Inhibition of clathrin-mediated endocytosis negatively regulates oocyte
maturation. (A) Membrane capacitance [Cm(nF)] of control
Xenopus oocytes and oocytes injected with C3 exoenzyme (1 ng/oocyte)
2 hours earlier. (B) GVBD timecourse from a representative experiment
after C3 exoenzyme injection. Cells were injected with C3 exoenzyme 1 hour
before progesterone treatment or left untreated. The histograms indicate
percent GVBD and normalized time to GVBD50 (mean±s.e.;
n=3). GVBD levels are those reached at the end of the experiment,
typically no longer than 18 hours. (C) Transferrin endocytosis assay.
Examples of confocal cross-sectional images through oocytes used to quantify
Alexa-633-labeled transferrin internalization after overnight incubation with
either the carrier control DMSO or 332 μM MDC. The number of labeled
vesicular structures and their equivalent sphere volumes were quantified, as
detailed in Materials and methods. (D) Membrane capacitance of oocytes
incubated in the DMSO carrier control and oocytes treated with 332 μM
monodansyl cadaverine (MDC) overnight. (E) GVBD time course from a
representative experiment after MDC treatment. Cells were incubated with MDC
overnight or left untreated before progesterone treatment. The histograms
indicate percent GVBD and normalized time to GVBD50
(mean±s.e.; n=5). Scale bar: 100 μm. Con, control
oocytes.

We next tested the effect of inhibition of the clathrin mediated endocytic
pathway using monodansylcadaverine (MDC)
(Schlegel et al., 1982). We
first developed a functional assay to confirm the ability of MDC to inhibit
clathrin-mediated endocytosis in Xenopus oocytes. We used
fluorescently labeled transferrin as a classical marker for clathrin-mediated
endocytosis (Dautry-Varsat,
1986), especially because Xenopus oocytes possess
functional transferrin receptors (Lund et
al., 1990). Although transferrin readily labels small endocytic
vesicles, the most reliable signal was from large vesicular structures, which
are potentially early endosomes (Fig.
5C). We quantified the levels of transferrin uptake using the
number and equivalent volume of these vesicular structures in a
cross-sectional confocal image (Fig.
5C). MDC effectively blocked transferrin endocytosis, as the
number of the presumed early endosomes detected in MDC-treated cells was
dramatically reduced (34.3±11.7% of control)
(Fig. 5C). Although the average
volume of labeled endosomes tended to be smaller in MDC-treated cells, the
data did not reach statistical significance
(Fig. 5C). These results show
that MDC blocks clathrin-mediated endocytosis in Xenopus oocytes and
can be used to test the role of this pathway on meiotic arrest.

In contrast to the C3 exoenzyme treatment
(Fig. 5A), MDC did not increase
membrane capacitance (Fig. 5D),
arguing that clathrin-dependent endocytosis has a much smaller contribution to
membrane area homeostasis compared with constitutive endocytosis. However,
blocking clathrin-dependent endocytosis with MDC slows down the rate and
inhibits the extent of progesterone-mediated maturation
(Fig. 5E). This shows that
inhibition of clathrin-mediated endocytosis negatively regulates the ability
of progesterone to relieve meiotic arrest. Similar results were obtained with
SNAP25Δ20-mediated maturation (not shown). Therefore, the endocytic
blockade experiments support the exocytic block data, as they both show that
vesicular recycling at the cell membrane is a crucial determinant of oocyte
meiotic arrest.

DISCUSSION

The prolonged arrest of oocytes in meiotic prophase awaiting oocyte
maturation is a fascinating biological problem that has been tackled for
several decades, yet the mechanisms involved remain obscure. Recent data
support the argument that GPCR signaling is crucial in maintaining meiotic
arrest (Mehlmann, 2005). In
this study we have addressed the role of vesicular trafficking at the cell
membrane in maintaining meiotic arrest. We have shown that blocking exocytosis
induces hormone-independent oocyte maturation that is normal in every aspect
tested. We used a dominant-negative SNAP25 to inhibit exocytosis, because
SNAP25 localizes to the cell membrane
(Gonzalo and Linder, 1998) and
is thus likely to block trafficking specifically at this subcellular
compartment. The fact that an exocytic block is sufficient to induce oocyte
maturation is consistent with the idea that continuous insertion of membrane
proteins, including possibly a constitutively active GPCR, is required for
meiotic arrest. Alternatively, the exocytic block data would also be
consistent with a potential autocrine mechanism for maintaining meiotic
arrest, where the oocyte secretes signals that act on cell-surface receptors
to maintain meiotic arrest.

The SNAP25Δ20 mutant induces a very efficient exocytic block, which
leads to a dramatic decrease in membrane surface area. By contrast, when we
used other interventions, such as injection of tetanus toxin or BoNT A to
inhibit exocytosis, we were not able to induce a significant decrease in
membrane capacitance or oocyte maturation. Nonetheless, BoNT A potentiates the
ability of sub-threshold levels of progesterone to induce maturation, arguing
that even mild inhibition of exocytosis has significant functional
consequences in terms of inducing maturation. This supports the argument that
a robust block of exocytosis is required to release the oocyte from meiotic
arrest. Consistent with this conclusion, blocking ER-to-Golgi transport with
brefeldin A, which ultimately leads to disruption of the Golgi and inhibition
of exocytosis, was reported to induce oocyte maturation in Xenopus
(Mulner-Lorillon et al.,
1995). Although the efficiency of brefeldin at inducing meiotic
arrest is poor compared with progesterone and SNAP25Δ20, it illustrates
the point that other interventions that significantly inhibit exocytosis can
release meiotic arrest.

Consistent with a crucial role for the exocytic pathway in maintaining
meiotic arrest, blocking clathrin-mediated but not constitutive endocytosis
negatively regulates the ability of progesterone to release meiotic arrest.
One interpretation of these data is that because activated GPCRs are
internalized through a clathrin-mediated pathway, inhibition of this
desensitization route will increase the number of active GPCRs at the cell
membrane, thus countering the effects of progesterone. However, direct
evidence for this hypothesis will have to await identification of the
constitutively active GPCR responsible for maintaining meiotic arrest in
Xenopus oocytes.

Recent mouse and rat data show that constitutively active GPCRs (GPR3/12)
are required for meiotic arrest (Hinckley
et al., 2005; Freudzon et al.,
2005). A similar mechanism could be functional in
Xenopus, especially because frog oocytes maintain meiotic arrest
after removal of the surrounding follicular cells, arguing for an
oocyte-autonomous mechanism for meiotic arrest. A membrane progesterone
receptor has been hypothesized for a long time in Xenopus to induce
meiotic maturation (Maller,
2001). Recently this receptor was cloned and demonstrated to
induce oocyte maturation through positive induction
(Zhu et al., 2003;
Ben Yehoshua et al., 2006).
This shows that it is not functioning as the constitutively active GPCR to
maintain high cAMP levels. Rather, it supports the argument that signaling
through this membrane progesterone receptor antagonizes a putative
constitutively active GPCR pathway that maintains meiotic arrest.

Physiological relevance of the exocytosis block

A gradual decrease in membrane surface area during Xenopus oocyte
maturation is well documented (Kado et
al., 1981; Machaca and Haun,
2000) and is due to an early block of exocytosis, which is crucial
for the formation of the fluid-filled blastocoele cavity during embryogenesis
(Muller, 2001). The
blastocoele is formed due to polarized vectorial transport of Na+
ions into the intercellular space by an epithelium that surrounds the embryo
(Muller, 2001). In
Xenopus the biogenesis of this polarized epithelium can be traced
back to the exocytosis block during oocyte maturation, which leads to
sequestration of most ionic transporters into an intracellular vesicular pool
(Muller and Hausen, 1995;
Muller, 2001). As the early
blastomeres rapidly divide, they incorporate these intracellular vesicles
containing ionic channels and transporters into their basolateral membranes.
The apical membrane of this epithelium is formed by the oocyte cell membrane,
which is devoid of most transporters, thus forming a polarized epithelium
around the embryo.

Additional morphological and functional evidence supports the early
exocytosis block during Xenopus oocyte maturation. At the
ultrastructural level, the decrease in surface area during oocyte maturation
is illustrated by the disappearance of microvilli, which are enriched in
oocytes but practically absent in eggs
(Campanella et al., 1984;
Gardiner and Grey, 1983).
Protein secretion is blocked early on in maturation, specifically between the
trans-Golgi network and the plasma membrane
(Colman et al., 1985;
Leaf et al., 1990), while
other intracellular trafficking events (such as ER to Golgi) are unaffected
(Leaf et al., 1990;
Ceriotti and Colman, 1989).
Therefore, an early exocytic block while endocytosis stays functional leads to
a dramatic decrease in membrane surface area during Xenopus oocyte
maturation. It is believed that oocytes employ this mechanism to stock
membranes and membrane proteins internally in preparation for the rapid cell
divisions in embryogenesis (Angres et al.,
1991; Gawantka et al.,
1992). The mechanisms by which progesterone blocks exocytosis are
unknown, but it is clear that vesicular trafficking at the cell membrane is
crucial not only for maintaining meiotic arrest but also for early
embryogenesis.

Acknowledgments

We are grateful to Michael Hollmann for the gift of the Xenopus
oocyte expression vector pSGEM, and to Paul Roche for the mouse
Snap25B clone. We also acknowledge the use of the Confocal Microscopy
Laboratory at the University of Arkansas for Medical Sciences, which is
supported by NIH GrantP20RR16460 (PI: Larry Cornett, INBRE, Partnerships for
Biomedical Research in Arkansas) and NIH/NCRR Grant S10RR19395 (PI: Richard
Kurten, “Zeiss LSM 510 META Confocal Microscope System”). This
work was funded by grant GM61829 from the NIH.

Gawantka, V., Ellinger-Ziegelbauer, H. and Hausen, P.
(1992). Beta 1-integrin is a maternal protein that is inserted
into all newly formed plasma membranes during early Xenopus embryogenesis.
Development115,595
-605.

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